Methods for Detecting B-Isox Precipitates or Captured Proteins as Biofluid Biomarkers

The present application claims priority benefit of U.S. Provisional Application No. 63/351,813, filed 13 Jun. 2022, the entire contents of which application are hereby incorporated herein by reference.

A method detects, and makes it possible to diagnose or treat, a human disease in a human subject by adding an isoxazole to an obtained biofluid sample to form a biofluid isoxazole composition in the biofluid sample, and detecting a presence of the biofluid isoxazole composition. Research identified additional biomarkers, which made it possible to detect, diagnose or treat, a human disease in a human subject by, with or without adding an isoxazole to an obtained biofluid sample, detecting the biomarker.

Although usable for other purposes, the methods relate to methods for differential diagnostics, real-time pathophysiology monitoring, presymptomatic diagnostics, and pharmacoresponse measurement of conformational diseases and proteinopathies, such as neurodegenerative diseases, diabetes, cancer, psychiatric disorders, and even aging.

Conformational diseases include more than 50 disorders caused by the accumulation of unfolded or misfolded proteins. Improper protein folding can lead to deposit amorphous aggregates, such as p-TDP-43 aggregates, or ordered amyloid fibrils, such as synuclein and tau inclusions (1).

Proteinopathies refer to pathologies caused by certain misfolded aggregation-prone proteins, such as synuclein, TDP-43 and tau. For example, synucleinopathies and tauopathy.

Amyotrophic lateral sclerosis (ALS) is a progressive, heterogeneous neuromuscular disease with limited treatment options (2). ALS degenerative symptoms are characterized by adult-onset progressive loss of primarily motor neurons in the cerebral cortex, brain stem, and spinal cord, consequently leading to progressive muscle weakness, atrophy and typical death 3 to 5 years after symptom onset. Although few ALS-causing gene, including SOD1, TDP-43and C9orf72, have been discovered for ALS and their proteins convert to misfold structure and form pathological inclusions, current treatments or diagnostics are still hampered by lack of definitive targets (3-7). Currently, the typical time to ALS diagnosis is 10-16 months from symptom onset. Misdiagnoses are a key factor leading to diagnostic delay. So far, no biomarker is available for sporadic ALS, which covers 90% patients with ALS.

Alzheimer's disease (AD) is the most common cause of memory loss and cognitive abilities (e.g., dementia) and may contribute to 60-70% of cases worldwide. AD brain is featuring of extracellular amyloid-β (Aβ) plaques and intraneuronal neurofibrillary (Tau) tangles (8). Both of abnormal structures are highly insoluble and stacked amyloid fibrils. Aβ plaque is derived from proteolytic processing of a transmembrane protein, the amyloid precursor protein (APP). APP is cleaved by β- and γ-secretases to produce Aβ peptide. Evidence has shown that excessive Aβ production induces neurotoxicity, triggering neuronal tangle formation, and neuron loss in the plaques deposited brain regions. This process is believed as the pathogenic mechanism in both familial and sporadic AD. The second risk factor in AD is the microtubule-associate protein Tau. Tau stabilizes microtubule network, regulates axonal integrity and axonal transport. Disruption of microtubule might be caused by the loss of function of Tau through aberrantly hyperphosphorylation. Hyperphosphorylated Tau isoforms as the main component of neurofibrillary tangles (NFTs) and neuropil threads observed in AD brain. At least 19 amino acids are phosphorylated and correlate with AD severity. Accordingly, high-abundance phosphor-Tau (pTau-217) isoform observed in cerebrospinal fluid (CSF) and plasma was used as an early diagnostic marker of AD.

Parkinson disease (PD) is a progressive neurodegenerative disorder primarily affected motor system, usually occurred of rigidity, hypokinesia and tremor. Non-motor symptoms also developed as the disease worsens, including cognitive changes. PD is caused predominantly by the loss of dopaminergic neurons in the substantia nigra (SN), a basal ganglia structure located in the midbrain. The signature pathology of the PD is lewy bodies (LBs) (9). Misfolded alpha-synuclein (α-Syn) as the major component of the LBs observed in sporadic PD; and that mutations in the α-Syn are associated with some rare familiar PD. The second risk factor for PD is the Leucine-rich repeat kinase 2 (LARRK2). LARRK2 gene mutation accounts for 5% of familiar PD as well as 3% of sporadic cases. LRRK2 harbored G2019 mutation is more susceptible to forming α-Syn inclusions that links LRRK2 with protein-misfolding pathology. Currently, the diagnostic process often takes over a year to complete, and currently no approved methods is available for early diagnostic and monitoring disease progression.

b-isox, biotinylated isoxazole (6-(5-(Thiophen-2-yl) isoxazole-3-carboxamido) hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanoate), is a small molecule known to precipitate RNA-binding proteins enriched in stress granules and RNA granules. Most of the proteins precipitated by b-isox contained low-complexity (LC) domain, which is responsible for the interaction with b-isox. The LC domains are intrinsic disordered structure and self-interactions. This novel type of self-interaction domain transiently forms a cross-β polymeric condensed phase to perform crucial biological processes, including DNA transcription and replication, chromatin remodeling, nuclear pore passage, signal transduction, synaptic transmission, and cytoskeleton regulation by homotypic-or heterotypic cross-β multimeric interactions. In contrast to pathological cross-β aggregate stacked in-register, the physiological cross-β multimers are loose and reversible (10, 11, 12, 13).

Currently, the cost-effective biomarker testing and noninvasive diagnostic techniques for detection of pathophysiology in clinical and presymptomatic stage of conformational diseases and proteinopathies, particularly neurodegenerative disease, are unmet medical need. The present invention addresses this need and other needs.

Methods for advances made in the differential diagnostics, real-time monitoring pathophysiology, pharmacoresponse analysis, presymptomatic diagnostics, and subtyping of neurodegenerative diseases by detecting the presence of b-isox precipitates or the level of b-isox-captured proteins from plasma and CSF of patients. Example of neurodegenerative diseases includes, but not limited to, ALS, AD, parkinson disease dementia (PDD), dementia with Lewy body (DLB), parkinson disease without dementia (PDnD), multiple system atrophy (MSA), spinal muscular atrophy (SMA), limbic predominant age related TDP-43 encephalopathy neuropathological change (LATE-NC) and frontotemporal dementia (FTLD).

Example of b-isox precipitated proteins for ALS diagnosis includes, but not limited to, p-TDP-43, SOD1, GR repeat protein (poly (GR)), GA repeat protein (poly(GA)), GP repeat protein (poly(GP)), carbonic anhydrase 1 (CA1), cluster of differentiation 14 (CD14), myosin light chain 12B (MYL12B), peroxiredoxin 2 (PRDX2), stomatin (STOM), profilin1 (PFN1), β-actin (ACTB), glucose transporter 1 (GLUT-1), survival of motor neuron 1 (SMN), and annexin A5 (ANXA5).

Example of b-isox precipitated proteins for PD diagnosis includes, but not limited to, p-TDP-43, CA1, CD14, PRDX2, STOM, ANXA5, synuclein, cellular adhesion molecule L1 like (CHL1), RUVB like AAA ATPase1 (RUVBL1), neural EGFL like 2 (NELL2), ankyrin-1 (ANK1), and neuronal cell adhesion molecule (NrCAM).

Example of b-isox precipitated proteins for AD diagnosis includes, but not limited to, amyloid beta, phospho-TDP-43, TDP-43, Tau, STOM, and ANK1.

Methods for advances made in presymptomatic diagnostic of SOD1 inherited and sporadic ALS by detecting the level of b-isox-captured GR repeat proteins from plasma and CSF of patients.

A method for detecting a human disease in a subject, comprises detecting a presence of biofluid isoxazole-precipitates in a biofluid sample of the subject. In some embodiments, the method of claim, wherein the isoxazole in the isoxazole-precipitates is biotin-isoxazole, (6-(5-(Thiophen-2-yl)isoxazole-3-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate) or its salt or an analog thereof.

In some embodiments, the human disease is Amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC) or frontotemporal dementia (FTLD).

In some embodiments, the biofluid is cerebrospinal fluid (CSF) or plasma.

A method for detecting a human conformational disease in a subject, comprises detecting a presence of biofluid isoxazole-captured proteins in a biofluid sample of the subject.

In some embodiments, the isoxazole is biotin-isoxazole (6-(5-(Thiophen-2-yl)isoxazole-3-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate) or its analogous.

In some embodiments, the human conformational disease is Amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC) or frontotemporal dementia (FTLD), or diabetes.

In some embodiments, the isoxazole-captured proteins are detected by a polypeptide.

In some embodiments, the polypeptide is (a) an antibody, or (b) an immunoglobulin chain, or a binding domain thereof which binds to the isoxazole-captured proteins. In some embodiments, the human conformational diseases is ALS, and wherein the polypeptide for detecting ALS is an antibody against SOD1, C9orf72 dipeptide repeats, PFN1, PRDX2, phospho-TDP-43, CA1, MYL12B, CD14, ANXA5, STOM, SMN, ACTB, or GLUT1. In some embodiments, the human conformational diseases is AD, and wherein polypeptide for detecting AD is an antibody against APP, phospho-TDP-43, TDP-43, Tau, STOM, or ANK1. In some embodiments, the human conformational diseases is PD, and wherein polypeptide for detecting PD is an antibody against synuclein, CHL1, NELL2, p-TDP-43, NrCAM, ANK1, STOM, PRDX2, CA1, CD14, and RUVBL1. In some embodiments, the biofluid is plasma and CSF.

A method for detecting conformational disease in a subject, comprises the step of detecting the biofluid level of proteins with cross-β structure in the sample of the subject.

In some embodiments, the biofluid is plasma and CSF. In some embodiments, the proteins with cross-β structure is detected by a combination of a cross-β recognized probe and a polypeptide. In some embodiments, the conformational disease is ALS, AD, PDD, PDnD, DLB, MSA, SMA, FTLD, diabetes, schizophrenia, cancer, aging and limbic predominant age related TDP-43 encephalopathy neuropathological change (LATE-NC) or diabetes.

The use of a reagent for detecting a b-isox captured complex in the manufacture of a kit to evaluate the risks of developing conformational disease or the progression of conformational diseases.

A method for detecting aging and conformational disease in a subject, comprises the step of detecting the presence of low complexity protein' complex in the sample of the subject.

A method for detecting sporadic ALS in a subject, comprises the step of detecting the presence of dipeptide repeat proteins in the sample of the subject.

In some embodiments, the dipeptide repeat protein is poly (GR), poly (GP), and poly (GA).

A method for detecting sporadic ALS and SOD1 inherited ALS at the presymptomatic and prodromal stage in a subject, comprises the step of detecting the presence of dipeptide repeat proteins in the sample of the subject.

In some embodiments, the dipeptide repeat protein is poly (GR).

The terms “invention,” “the invention,” “this invention” and “the present invention” used in this patent are intended to refer broadly to all of the subject matter of this patent and the patent claims below. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the patent claims below. Embodiments of the invention covered by this patent are defined by the claims below, not this summary. This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are further described in the Detailed Description section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all drawings and each claim.

The invention will become more apparent when read with the accompanying figures and detailed description which follow.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) of the invention and together with the description, serve to explain the principles of the invention.

Reference will now be made in detail to the present embodiment(s) (exemplary embodiments) of the invention, an example(s) of which is (are) illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

Methods for the detection of conformational diseases and proteinopathies, such as neurodegenerative diseases, are described herein by using a small-molecule to generate visual precipitates.

In addition, applicants identified novel and specific biofluid biomarkers for differential diagnosis and monitoring pathophysiology in patients with neurodegenerative diseases, including ALS, AD and PD.

Also described are novel methods for the plasma diagnosis of neurodegenerative diseases. The novel method named b-isox-ELISA, is combination of b-isox chemical-precipitation and immunoassay with specific biomarkers of ALS, AD or PD. b-isox ELISA can be used to screen the risks of ALS, AD and PD. This invention can be used to distinguish between AD, TDP-43 proteinopathies, PD, and dementia with Lewy bodies, ALS subtyping, real-time readout of pharmacoresponse, and monitoring the relief of pathological burden of misfolded disease proteins in clinical trial, preclinical diagnosis and clinical practice.

As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.

Embodiment 1. A method for detecting a human disease in a human subject, comprises, optionally obtaining a biofluid sample from the subject, adding an isoxazole to the obtained biofluid sample to form a biofluid isoxazole composition in the biofluid sample, and detecting a presence of the biofluid isoxazole composition.

Embodiment 2. The method of Embodiment 1, wherein the isoxazole is biotin-isoxazole, (6-(5-(Thiophen-2-yl)isoxazole-3-carboxamido)hexyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), or its salt or an analog thereof, especially biotin-isoxazole or its salt and very especially biotin-isoxazole.

Embodiment 3. The method of Embodiments 1-2, wherein the biofluid sample is urine, whole blood, plasma, or serum, cerebrospinal fluid (CSF), saliva, or mucosa, such as, urine, saliva, CSF or plasma, and especially CSF or plasma. In some embodiments, the biofluid sample is in the form of a biological fluid such as urine, whole blood, plasma, or serum, cerebrospinal fluid (CSF), saliva, or mucosa, and optionally, the biofluid sample is further processed, e.g., to remove some components, e.g., by techniques to enrich components such as proteins by chemical precipitation. In some embodiments, the biofluid sample is blood, plasma, or serum, CSF, urine or saliva. In some embodiments, the biofluid sample is plasma or CSF.

Embodiment 4. The method of Embodiments 1-3, wherein the human disease is Amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), frontotemporal dementia (FTLD), diabetes, cancer, infectious disease, huntington disease, schizophrenia, aging-associated disease, or protienopathies, especially wherein the human disease is Amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

Embodiment 5. The method of Embodiments 1-4, further comprises thereafter treating the human disease or thereafter changing an existing treatment based on the detecting the presence of the biofluid isoxazole composition, such as a treatment including pharmaceutical therapy for the human disease. In some embodiments, the method further comprises diagnosing the human disease.

Embodiment 6. The method of Embodiments 1-5, wherein the concentration of isoxazole ranges from 0.075 mM to 0.225 mM, preferably from 0.100 mM to 0.200 mM, in the biofluid sample.

Embodiment 7. The method of Embodiments 1-6, wherein the biofluid isoxazole composition is in a precipitate.

Embodiments 8. The method of Embodiment 7, wherein the human disease is Amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), parkinson disease dementia (PDD), parkinson disease no dementia (PDnD), dementia with Lewy body (DLB), multiple system atrophy (MSA), spinal muscular atrophy (SMA), and limbic predominant age related TAR DNA-binding protein 43 (TDP-43) encephalopathy neuropathological change (LATE-NC), stroke, cerebral amyloid angiopathy (CAA), or frontotemporal dementia (FTLD).

Embodiment 9. The method of Embodiment 8, wherein the human disease is ALS, AD, PDD, DLB, MSA or PDnD.